Huntington""s Disease (HD) is an autosomal dominant neurodegenerative disorder that causes progressive motor and cognitive impairments in its victims, ultimately leading to death (Wexler et al., Ann. Rev. Neurosci. 14:503-529, 1991; Folstein, In Huntington""s Disease: A Disorder of Families. Johns Hopkins University Press, pages 1-64, 1989). Although widespread abnormalities in the HD brain have been reported, the striatum is the most severely affected structure, exhibiting marked atrophy and loss of neurons, particularly of medium-sized spiny cells (Graveland et al., Science 227:770-773, 1985; Vonsattel et al., J. Neuropath. Exp. Neurol. 44:559-577, 1985).
The link between the genetic change in HD and processes leading to cell death remain elusive. The gene associated with HD, IT15 (for interesting transcript 15), was isolated (The Huntington""s Disease Research Collaborative Group, Cell 72:971-983, 1993) and shown to predict a 348 kDa protein, huntingtin. The HD-causing mutations in IT15 involve an expansion of a trinucleotide (CAG) repeat encoding glutamines near the 5xe2x80x2 end of the coding sequence. In general, individuals with 37 or more repeats will get Huntington""s disease. IT15 bears no homology to other known genes, and its function in disease is unknown.
The normal biological role of wild-type huntingtin is also unknown. This role is presumed to be important because deletion of both huntingtin genes in the mouse leads to death on about the seventh embryonic day (E7), before the brain is formed (Duyao et al., Science 269:407-409, 1995).
Huntingtin is found in soluble and membrane-associated compartments in cells. In synaptosomal membrane fractions of the HD brain, mutant huntingtin is apparently present at levels comparable to the wild-type protein (Aronin et al., Neuron 15:1193-1201, 1995; Aronin et al., J. NIH Res. 7:55-56, 1995). This observation implies that both proteins are anterogradely transported and associated with the same intracellular membranes. A major obstacle to identifying the potential sites of protein interactions of mutant huntingtin is distinguishing its subcellular location from that of the wild-type protein. This cannot be done in brain, where only heterozygote tissue is currently available.
As described herein, the subcellular distribution of wild-type and mutant huntingtin overlaps that of clathrin. Thus, wild-type huntingtin may play a critical role in the process of clathrin-mediated endocytosis. Furthermore, when a mutant form of huntingtin is expressed, as occurs in Huntington""s Disease, the characteristic impairment and eventual death of neurons that ensues may be caused by a defective endocytotic process. The invention involves identifying compounds that may remedy defects in the endocytotic process associated with the presence of mutant huntingtin.
The invention features a method of identifying a compound useful in the treatment of a disease characterized by the presence of mutant huntingtin by: (a) providing a cell which expresses mutant huntingtin; (b) contacting the cell with a test compound; and (c) determining whether the level of clathrin-mediated endocytosis is increased in the presence of the test compound compared to the level in the absence of the test compound. An increase in clathrin-mediated endocytosis is an indication that the test compound is useful in treating the disease.
The test compound can be a compound that activates either GTP-binding proteins or protein kinase C (e.g., a phorbol ester). Alternatively, the test compound can be a compound that alters the pH (e.g., raising the pH by at least 0.5 pH units, and preferably by 1.0 pH units) or the concentration of calcium in the extracellular environment.
Cells to be used in the invention can be obtained from a patient who has been diagnosed as having Huntington""s Disease. The presence of 37 or more of the CAG repeats in the huntingtin gene are diagnostic of Huntington""s Disease. Because the disease is dominant, a person need only have one copy of the gene to get Huntington""s Disease. Furthermore, the disease is virtually 100% penetrant. Therefore, a person diagnosed as having Huntington""s disease is a person who has an expanded CAG repeat region in at least one of their huntingtin genes although that person may have no or few symptoms of the disease at the time of diagnosis. Alternatively, the cells may be any cells that express mutant huntingtin including cells that have been genetically engineered. Preferably, the cells are fibroblasts. More preferably, the cells are neurons.
The level of clathrin-mediated endocytosis can be determined by monitoring the amount of a substance taken up from the extracellular medium in a given amount of time. This can be measured, for example, by monitoring the uptake of biotinylated transferrin (b-transferrin) and its receptor, as described herein, and may reflect a change in the rate of an endocytotic step.
The invention also features a method of determining whether a compound is useful for treatment of a patient who has been diagnosed as having Huntington""s Disease. In practicing this method, one would obtain appropriate cells from the patient, contact those cells with the compound, and determine whether the level of clathrin-mediated endocytosis is increased in the presence of the compound, compared to the level in a control sample in the absence of the compound. An increase in clathrin-mediated endocytosis is an indication that the compound is useful in treating the patient.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. These methods, materials, and examples are illustrative only and not intended to be limiting.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.